As the parasite genome sequencing projects approach completion, thousands of new genes are being identified. However, fewer than half have ascribed functions, largely based on sequence homology, and the function of a substantial proportion of the remainder is only poorly understood. If this potential wealth of this data is to be fully utilized for the development of novel parasite control strategies, new paradigms for carrying out functional studies on parasite genes must be developed and implemented. The emphasis must be shifted from the traditional "gene-by-gene" approach to a more global strategy for identifying genes with crucial parasite functions to study in more detail. We propose to use such an approach to investigate the molecular processes underlying differentiation between the insect (promastigote) and mammalian (amastigote) stages of Leishmania. This represents a critical time-period during the parasite lifecycle and presents an excellent opportunity for therapeutic intervention. We hypothesize that there is an ordered progression of specific changes in gene expression during this transition, and that genes whose expression changes early in this transition regulate those whose expression changes later. It is likely to have sequence similarity/homology to those involved in signal transduction and heat shock response pathways in other organisms. We will test this hypothesis by utilizing several high-throughput genome-wide approaches to identify genes whose expression is regulated during the promastigote-to amastigote transition. Since Leishmania regulates gene expression at the level of both mRNA and protein abundance, we will use techniques designed to detect differences at both levels. We will use techniques that will identify genes who's differential expression is regulated at the level of mRNA abundance (SAGE and DNA micro array screening) and/or regulated at the level of protein abundance (2-D gel electrophoresis and mass spectrometry). We will use an integrated Biolnformatics system to prioritize those genes likely to be encoding proteins with regulatory functions (e.g., those whose expression changes early in the transition, in response to changes in pH and temperature), and use reverse genetic approaches to confirm their importance and elucidate their function. These will include gene replacement to obtain null mutants, and over-expression using a regulatable promoter system. These experiments will also provide a model for more comprehensive studies to elucidate the molecular biology of Leishmania and other trypanosomatids.